Depression and anxiety affect 3 to 5% of the US population and these conditions frequently affect the same individuals. Evidence suggests that serotonin (5HT) neurons play a role in depression and anxiety, and SSRIs such as fluoxetine and other medications that target the 5HT system are available. However, the existing treatments are ineffective for many drug-resistant patients. The habenula provides input to the 5HT system, located in the raphe, from higher brain centers. It can be divided into the medial (MHb) and lateral habenula (LHb), and each region contributes to distinct pathways. While the habenula is known to be involved in many behavioral functions, including stress and depression, previous studies have not distinguished between the MHb and LHb. In the proposed studies, requesting postdoctoral fellowship support for Dr. Yun- Wei Hsu, we ask 1) do the dorsal and ventral medial habenula (MHb) serve distinct downstream pathways in the interpeduncular nucleus and the midbrain 5HT system? 2) Does genetic lesion of dorsal MHb neurons decrease anxiety and depression-like behaviors? This project will target specifically the MHb and will investigate the pathway between the MHb and the 5HT neurons that mediate depression using a combination of genetic and optogenetic strategies. In our first approach, light-sensitive channels (Channelrhodopsin-2) will be expressed in neurons in different MHb regions using a combinatorial genetic system developed in collaboration with the Allen Brain Institute. The MHb neurons will be activated by light while the responses of the 5HT neurons in brain slice preparations will be recorded. This will determine how the MHb-raphe pathway is regulated. Neuronal activation using targeted optogenetics will allow the function of the different neuronal populations in the MHb to be distinguished for the first time. In our second approach, we will use cre-lox mediated excision of Brn3a, a transcription factor essential for habenula development, to generate mice that have a defect in the dorsal MHb. The pathway from the MHb to the downstream 5HT system will thus be disrupted in these mice. They will be tested in different behavioral paradigms that model anxiety and depression, including the open field test and the elevated plus maze for anxiety and the forced swim test for depression-like behaviors. These experiments will reveal the function of a poorly understood brain pathway that may lead to alternative treatments for drug-resistant patients suffering from depression and anxiety. The training program outlined in this application would provide professional development opportunities, allow the applicant to train with leading experts in electrophysiology and behavioral methods, and enhance writing and presentation skills, while building a foundation for a future independent research career.